Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
  • B01D15/3804—Affinity chromatography
  • B01D15/3828—Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
  • B01J20/26—Synthetic macromolecular compounds
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
  • B01J20/26—Synthetic macromolecular compounds
  • B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J45/00—Ion-exchange in which a complex or a chelate is formed; Use of material as complex or chelate forming ion-exchangers; Treatment of material for improving the complex or chelate forming ion-exchange properties
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

• the present invention relates, inter alia, to the field of isolation and purification of peptides, notably polypeptides, such as recombinant proteins, by means of immobilized hard metal ion affinity chroma- tography.
• peptides including oligo- and polypeptides, notably proteins, intended for therapeutic use in humans or animals
• purification of the peptides in question to a sufficiently high level of purity, such that the desired protein is essentially completely free of contamination with, in particular, (a) any extraneous proteins which may arise in the production process (typically a fermentation process or the like employing a selected or genetically modified strain of an appropriate microorganism) and (b) undesirable metal ions (notably heavy-metal ions) that may have been introduced in the course of the production process.
• Immobilized metal ion affinity chromatography is a versatile separation procedure that exploits differences in the affinities exhibited by many biopolymers for metal ions.
• the technique involves the chelation of a suitable metal ion onto a solid support matrix whose surface has previously been chemically modified with a polydentate ligand.
• the resulting immobilized metal ion chelating complex then has the potential to coordinate with one or more electron donor groups resident on the surface of the interacting protein (Sulkowski, E., Trends in Biotechnology, 3 (1985) 1-6; Porath, J., Carlsson, I., Ols- son, I.
• transition metal ions of borderline hardness such as Cu 2+ , Zn 2+ and Ni 2+ .
• These metal ions demonstrate intermediate metal ion stability constants, e.g. log ⁇ values be- tween 5 and 10, for both aromatic and aliphatic amines, as well as for carboxylate functional groups (Wong, J. W., Albright, R. L and Wang, N. H. L, Separation and Purification Methods, 20 (1991) 49- 57; Zachariou, M., Traverso, I.., Spiccia, L.
• unconstrained tridentate chelates that exhibit these binding proper- ties with M 2+ ions can be chemically immobilized onto support materials.
• unconstrained types of chelating compounds such as iminodiacetic acid (IDA) constitute the principal types of chelating ligand employed hitherto in such IMAC investigations [see, e.g., Kage- dal, L, in "Protein Purification” (Eds. J. C. Janson and L. Ryden), VCH Publishers (1989) pp 227-251].
• NTA being a structural homologue of IDA
• sorbent and "adsorbent” are used primarily to denote a functionalized polymer substrate (polymer substrate with ligand immobilized thereto) with coordinatively bound metal ion(s), although these terms are also occasionally employed to denote a functionalised polymer substrate without metal ion(s) bound thereto.
• CM-ASP carboxymethylaspartic acid
• orffto-phosphoserine which is able to chelate "hard” metal ions such as Fe 3+ , Al 3+ , Ca 2+ and Yb 3 " due to the participation of the phosphate group [Zachariou, M., Traverso, I. and Hearn, M. T.
• NTA nitrilotriacetic acid
• N,N,N'-tris(carboxymethyl)ethylene-diamine [Porath, J., Protein Expression & Purification, 3 (1992) 263-281], which coordinate metal ions via five donor atoms (i.e. two nitrogen atoms of primary amine groups and three nitrogen atoms of secondary amine groups in the case of TEPA, and two nitrogen atoms of secondary amine groups and three oxygen atoms from the three carboxylic groups in the case of TED).
• WO 03/042249 relates, inter alia, to classes of functionalized polymer substrates containing a functionality comprising one or more cyclic, metal ion coordinating ligand groups having at least 3 metal ion coordinat- ing donor atoms chosen independently among N, O and S.
• these functionalized polymer substrates When employed as a matrix for one or more metal ions that form(s) coordination bonds to these donor atoms whilst retaining vacant coordination sites, these functionalized polymer substrates were found to exhibit remarkably high strength and/or selectivity of binding towards fusion proteins in the form of proteins or polypeptides "tagged” with an additional oli- gopeptide sequence ('tag") incorporating one or more appropriately positioned amino acid residues ca- pable of forming a coordination bond to the vacant coordination site(s) of the metal ion or ions in question.
• 'tag additional oli- gopeptide sequence
• Preferred functionalized (metal ion coordinating) polymer substrates disclosed in WO 03/042249 employ functionalities in which the metal ion coordinating donor atoms in each cyclic, metal ion coordinating group consist of three nitrogen donor atoms in the ring, and they are particularly well suited for use as a matrix for certain metal ions of borderline properties with respect to "hardness” or "softness” (vide infra), such as Cu 2+ or Ni 2+ .
• One objective of the present invention was to provide novel IMAC systems based on "hard” metal ions (such as Ca 2+ , Mg 2+ and Fe 3+ ) and metal ions at the "hard” end of the scale with respect to "borderline” hardness (such as Zn 2+ ) that function through interactions with hard donor atoms [especially oxygen atoms in carboxylate groups (as in Asp or Glu amino acid residues) and/or phosphate groups] present in biomolecules.
• An important feature of these novel IMAC-based systems is that they can concur- rently achieve a mixed modality of interaction with their target molecules that is based on a combination of coordination (electron donor/electron acceptor) and electrostatic (ion-exchange) processes. As a consequence, these novel IMAC-based systems can function with selectivity mediated through mixed modes of interaction that are unique and thus offer the opportunity for a new capability in protein purification.
• One aspect of the present invention thus relates to a polymer substrate functionalized with a functionality comprising at least one cyclic, metal ion coordinating ligand group which comprises at least 3 nitrogen donor atoms in the ring of said cyclic group, at least one of said nitrogen atoms having an optionally substituted carboxy(lower alkyl) group or an optionally substituted phosphono(lower alkyl) group covalently attached thereto.
• a second aspect of the invention relates to a functionalized polymer substrate of the latter type, further comprising a metal ion coordinated to at least one of the cyclic ligand groups in the functionality.
• Other aspects of the invention include methods for preparing such functionalized polymer substrates.
• fusion proteins of the type in question comprising a protein of interest fused at its amino terminus or carboxy terminus or both, or alternatively at a location within the internal amino acid sequence of the protein of interest, to at least one such oligopeptide;
• polynucleotide constructs e.g. vectors, encoding such fusion proteins
• a method for producing a fusion protein of the type in question wherein a host cell of the latter type is cultivated in a growth medium under conditions whereby the fusion protein is expressed, and whereby the fusion protein is recovered from the medium; and a method for purifying a protein of interest, wherein a wild-type protein or a protein sample containing such a fusion protein (comprising the protein of interest) as well as other proteins (extraneous proteins) is contacted with a functionalized polymer substrate according to the invention or a metal ion- containing functionalized polymer substrate according to the invention.
• a first aspect of the invention relates to a polymer substrate functionalized with a functionality comprising at least one cyclic, metal ion coordinating ligand group which comprises at least 3 nitrogen donor atoms in the ring of the cyclic group, at least one of the nitrogen atoms having an optionally substituted carboxy(lower alkyl) or optionally substituted phosphono(lower alkyl) group covalently attached thereto.
• Useful polymer substrates in the context of the invention include both water-soluble polymers and substantially water-insoluble polymers, and may be selected from a very wide range of polymeric materials. Examples hereof are the following:
• Polysaccharides and derivatives thereof including agaroses, dextrans, celluloses, hemicelluloses, starches, xylans and the like, and derivatives of these polysaccharides.
• Suitable polysaccharide derivatives will, in general, include derivatives in which some proportion of the hydroxy groups of the polysaccharide in question is derivatized to form ethers (e.g. lower alkyl ethers, such as methyl ethers) or esters (e.g. lower carboxylic acid esters, such as acetate, propionate and the like), as well as mate- rials in which the starting polysaccharide or a derivative thereof has been cross-linked by treatment with an appropriate cross-linking reagent.
• ethers e.g. lower alkyl ethers, such as methyl ethers
• esters e.g. lower carboxylic acid esters, such as acetate, propionate and the like
• functionalized polymer substrates of the invention based on substantially water- insoluble polymers are, for example, well suited for packing into chromatography columns, for direct introduction into a medium (batchwise use) and the like, and polysaccharides that are particularly well suited for this type of application in the context of the invention include agaroses, dextrans and derivatives thereof, a variety of suitable types of which are readily commercially available.
• agaroses are produced by Amersham Pharmacia Biotech, Uppsala, Sweden, and marketed under the name SepharoseTM; available grades include SepharoseTM 2B, 4B and 6B.
• Cross- linked derivatives of these various grades of agarose are also available from the same company, and are marketed as SepharoseTM CL-2B, CL-4B and CL-6B, SepharoseTM 4 and 6 Fast Flow, SepharoseTM 6MB, and SuperoseTM 6 and 12, respectively.
• SepharoseTM CL-2B, CL-4B and CL-6B SepharoseTM 4 and 6 Fast Flow
• SepharoseTM 6MB SepharoseTM 6MB
• SuperoseTM 6 and 12 are also available from Amersham Pharmacia Biotech under the names SephadexTM, SuperdexTM (e.g. SuperdexTM 30, 75 and 200) and SephacrylTM.
• Products in the SephadexTM range are prepared by cross-linking dextran with epichlorohydrin and are available in the following grades: SephadexTM G-10, G-15, G-25, G-50, G-75, G-100, G-150 and G-200, the degree of cross-linking decreasing with increasing G number.
• Products in the SephacrylTM range are prepared by cross-linking allyl-dextran with W,/V-methylene-bisacrylamide, and include SephacrylTM S-100, S-200, S-300, S-400, S-500 and S-1000; the latter six products differ with respect to their range of pore size and particle size distribution.
• Products in the SuperdexTM range are prepared by cross-linking allyl- dextran with agarose derivatives of various compositions.
• Polvalkylene ⁇ lvcols and derivatives thereof including, in particular, polyethylene glycols (PEG), i.e. condensation polymers of ethylene glycol having the general formula HOCH 2 (CH 2 OCH2) ⁇ CH2 ⁇ H or H(OCH 2 CH 2 ) ⁇ OH and typically having average molecular weights in the range from 200 to 6000.
• PEG polyethylene glycols
• a number of PEG'S (including PEG'S of average molecular weight 400, 600, 1500, 4000 and 6000, respectively) are available under various names (e.g. MacrogolTM, PEGTM, CarbowaxTM, NycolineTM, Plu- racol ETM, Poly-GTM, Polyglycol ETM, SolbaseTM) from a variety of commercial sources.
• PEG's are generally soluble in or miscible with water, as well as in ethanol and a number of other organic solvents, including aromatic hydrocarbons.
• the analogous polypropylene glycols [having the general formula H(OC 3 H 6 ) ⁇ OH], the lower molecular weight members of which are soluble in water, are also of relevance in the context of the invention.
• Relevant derivatives of such polyalkylene glycols include partially etherified derivatives, e.g. derivatives in which one of the terminal hydroxy groups has been converted to a lower alkyl ether group, such as a methyl ether group.
• Such polymers can readily be immobilized to support materials, thereby producing substrates that can subsequently be activated and then functionalized or derivatized with macrocyclic metal ion binding chelating ligands by procedures according to the present invention.
• Polwinyl polymers including polyvinyl alcohols - i.e. hydroxylic polymers normally produced by hy- drolysis ("alcoholysis") of various molecular weight fractions of polyvinyl acetate, typically by base or acid hydrolysis - and derivatives thereof.
• the degree of "alcoholysis” may be varied by either allowing the hydrolysis of acetate ester groups in polyvinyl acetate to proceed to substantial completion, or by stopping it at a desired degree of alcoholysis.
• Polyvinyl alcohols are normally commercially available in four molecular weight ranges, viz. ca. 250,000-300,000 (termed super-high viscosity), ca. 170,000-ca.
• polyvinyl alcohols within all of the above-outlined categories are or relevance in the context of the present inven- tion, as are, for example, ether derivatives thereof, such as methyl ether derivatives.
• polyvinyl polymer materials of interest include materials such as the ToyopeariTM HW range of porous, semi-rigid spherical gel particles designed for medium- and low-pressure liquid chromatogra- phy. Such materials, after activation and functionalization/derivatization, provide another option for the preparation of IMAC sorbents of relevance in the context of the invention.
• ToyopeariTM HW gels (obtainable from Tosoh Corp, Yamaguchi, Japan, and other suppliers) are synthesized from hydrophilic vinyl polymer containing exclusively C, H and O atoms.
• Available grades include ToyopeariTM HW-40, HW-40C, HW-40F, HW-40S, HW-50, HW-50F, HW- 50S, HW-55, HW-55F, HW-55S, HW-65F, HW-65S and HW-75F.
• Pol vacrylam ides and derivatives thereof including composite materials based on polyacrylamide and agarose, such as UltrogelTM AcA gels (composite polyacrylamide-agarose gel in bead form, available from, e.g., Amersham Pharmacia Biotech).
• UltrogelTM AcA gel range includes AcA 22, AcA 34, AcA 44 and AcA 54, where the number refers to the percentage of acrylamide and agarose, i.e., AcA 22 contains 2% acrylamide and 2% agarose.
• Activation of hydroxylic groups of these support materials provides an avenue to the preparation of IMAC sorbents.
• glycidylpropoxy-modified porous silica such as LiChroSpherTM Diol (E. Merck, Darmstadt, Germany), ToyosodaTM TSKSW3000 (Tosoh Corp., Yamaguchi, Japan); amino- propyl-modified silica, prepared by reaction (in the presence of a suitable catalyst) of aminopropyldi- ethoxysilane with silicas of appropriate pore size and appropriate average diameter; and mercapto- propylsilicas, prepared by reaction (in the presence of a suitable catalyst) of mercaptopropyldiethoxysi- lane with silicas of appropriate pore sizes and appropriate average diameters.
• glycidylpropoxy-modified porous silica such as LiChroSpherTM Diol (E. Merck, Darmstadt, Germany), ToyosodaTM TSKSW3000 (Tosoh Corp., Yamaguchi, Japan
• dextran modified or butadiene-vinyl copolymer modified silicas of appropriate pore sizes and appropriate aver- age diameters can be employed as the chromatographic support materials.
• "Naked" porous silicas suitable for such derivatization and subsequent modification to generate the respective novel IMAC sorbents can readily be obtained from a variety of suppliers, including E. Merck, (Darmstadt, Germany), Tosoh Corporation, Yamaguchi, Japan), Eka-Nobel AB (G ⁇ teborg, Sweden) and Grace Davi- son GmbH (Worms, Germany).
• Surface-modified metal oxides including glycidylpropoxy-modified porous zirconias, titanias or aluminas, as well as modifications/variants thereof based on the respective metal oxide "doped" with a second metal oxide; amino-propyl-modified zirconia, titania or alumina, prepared by reaction (in the presence of a suitable catalyst) of aminopropyldiethoxysilane with the zirconia, titania or alumina of appropriate pore size and appropriate average diameter; and mercaptopropyl-modified zirconia, titania or alumina, prepared by reaction (in the presence of a suitable catalyst) of mercaptopropyldiethoxysi- lane with the zirconia, titania or alumina of appropriate pore size and appropriate average diameter.
• dextran modified or butadiene-vinyl copolymer modified zirconia, titania or alumina of appropriate pore sizes and average diameters can be employed as the chromatographic support ma- terials.
• "Naked" porous zirconia, titania or alumina suitable for such derivatization and subsequent modification to generate the respective novel IMAC sorbents can readily be obtained from a variety of suppliers, including YMC Co. Ltd. (Kyoto, Japan), Grace GmbH (Worms, Germany) and BioSepra Corp. (Paris, France).
• Well suited polymer substrates in the context of the invention include agaroses, dextrans and derivatives thereof, e.g. materials selected among those outlined above.
• the cyclic, metal ion coordinating ligand group in a functionalized polymer substrate according to the invention is derived from a heterocycle chosen among: triazacycloal- kanes and -cycloalkenes; and tetraazacycloalkanes and -cycloalkenes.
• a heterocycle chosen among the following:
• the optional substituent(s) on the lower alkyl group, lower alkoxy group or aryl group in question may optionally comprise one or more metal ion coordinating donor
• lower alkyl as employed in the context of the present invention in intended to designate any linear (straight-chain), branched or cyclic alkyl group having from 1 to 6 carbon atoms.
• linear alkyl groups are methyl, ethyl, propyl, butyl, pentyl and hexyl;
• branched alkyl groups are isopropyl, iso-butyl, sec-butyl, tert-butyl, isopentyl and isohexyl;
• examples of cyclic alkyl groups are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
• linear or branched lower alkyl groups having from 1 to 3 carbon atoms i.e. methyl, ethyl, propyl and isopropyl
• Suitable optional substituents on lower alkyl groups in the context of the invention include halogen, hydroxy, lower alkoxy and optionally substituted aryl.
• lower alkoxy as employed in the context of the present invention in intended to designate any linear, branched or cyclic alkoxy group having from 1 to 6 carbon atoms.
• linear alkoxy groups are methoxy, ethoxy, propoxy, butoxy, pentoxy and hexoxy;
• examples of branched alkoxy groups are isopropoxy, sec-butoxy, tert-butoxy, isopentoxy and isohexoxy;
• examples of cyclic alkoxy groups are cyclopropyloxy, cyclobutyloxy, cyclopentyloxy and cyclohexyloxy.
• linear or branched lower alkoxy groups having from 1 to 3 carbon atoms i.e. methoxy, ethoxy, propoxy and isopropoxy
• aryl is intended to designate any aromatic group and includes both carbocyclic and heterocyclic aromatic groups. Examples thereof are phenyl, naphthyl, pyridyl, tetrazolyl, thiazolyl, imidazolyl, indolyl, quinolinyl, pyrimidinyl, thiadiazolyl, pyrazolyl, oxazolyl, isoxazolyl, thienyl, furanyl or oxadiazolyl.
• Suitable optional substituents on aryl groups in the context of the invention include halogen, amino, hydroxy, lower alkyl and lower alkoxy.
• halogen designates Cl, F, Br or I.
• optionally substituted carboxy(lower alkyl) or optionally substituted phos- phono(lower alkyl) group covalently attached to at least one of the ring N atoms of the cyclic metal ion coordinating ligand group, carboxymethyl (-CH 2 COOH) and phosphonomethyl [-CH 2 PO(OH) 2 ], respec- tively, have proved to be very suitable.
• metal ion coordinating ligand groups of the triaza- cycloalkane or -cycloalkene type mentioned above, it appears to be advantageous that at least two (i.e.
• two or three) of the three ring N atoms have an optionally substituted carboxy(lower alkyl) or optionally substituted phosphono(lower alkyl) group (e.g. a carboxymethyl or phosphonomethyl group) covalently attached thereto.
• metal ion coordinating groups of the tetraazacycloalkane or -cycloalkene type mentioned above, it appears to be advantageous that at least two (i.e. two, three or four) of the four ring N atoms have an optionally substituted carboxy(lower alkyl) or optionally substituted phosphono(lower alkyl) group (e.g. a carboxymethyl or phosphonomethyl group) covalently attached thereto.
• Suitable optional substituents on the lower alkyl moiety in such optionally substituted carboxy(lower alkyl) or optionally substituted phosphono(lower alkyl) groups include optionally substituted aryl, i.e. aryl (e.g. phenyl) and substituted aryl [e.g.
• lower alkylphenyl such as methylphenyl, ethylphenyl, propylphenyl, isopropylphenyl, cyclopropylphenyl, cyclobutylphenyl, cyclopentylphenyl or cyclohexylphenyl, where the lower alkyl group on the phenyl ring may be in any position (i.e. 2-, 3- or 4- position) relative to the carbon atom bearing the carboxy group or phosphono group.
• linker or spacer group X may be any suitable type of linker or spacer, but will typically be one which may be derived from a bifunctional organic compound (e.g.
• a type of linker or spacer group X which is generally very useful in the context of the present invention is one which can be derived from epichlorohydrin by reaction of the halogen end thereof with, e.g., an hydroxy group on the surface of the polymer substrate in question and then reaction of the epoxy group thereof with a substituted amino group in a cyclic ligand group.
• the linker or spacer group X is a group derivable from epichlorohydrin by reaction thereof with the polymer sub- strate in the form of an agarose or agarose derivative, and subsequent reaction of the resulting product with a ring -NH- group of the cyclic, metal ion coordinating ligand group which becomes bound to X.
• an important feature of the materials (functionalized polymer substrates) employed according to the invention to isolate and purify a desired protein (protein of interest) is the presence, in the material, of a metal ion which itself is bound coordinatively to a cyclic ligand group in the functionality, and which in turn is capable of binding coordinatively, and suitably selectively, to donor atoms in the amino acid residues of the oligopeptide "tag" part of a fusion protein in which the oligopeptide 'tag" is attached to the amino acid sequence of the protein of interest.
• a fur- ther aspect of the invention thus relates to a functionalized polymer substrate as described above, in which at least one of the cyclic ligand groups in the functionality has a metal ion coordinated thereto.
• Functionalized polymer substrates disclosed and described herein are particularly well suited to coordination of certain divalent (2+-charged) or trivalent (3+-charged) metal ions, notably metal ions chosen among Ca 2+ , Mg + , Zn 2+ and Fe 3+ .
• Ca 2+ is a versatile metal ion in this connection.
• a further aspect of the invention relates to a process for preparing a functionalized polymer substrate according to the invention, the process comprising the steps of: selecting a polymer substrate having a reactive functional group capable of undergoing a first reaction with a first functional group of a bifunctional reagent having a first and a second functional group; the first reaction in question resulting in covalent bond formation between the polymer substrate and the bifunctional reagent; the second functional group of the resulting covalently bound reagent being sub- sequently capable of undergoing a second reaction with a reactive ring -NH- group present in a species comprising at least one cyclic, metal ion coordinating ligand group which comprises at least 3 nitrogen donor atoms in the ring of the cyclic group, at least one of the nitrogen atoms in question having an optionally substituted carboxy(lower alkyl) or phosphono(lower alkyl) group covalently attached thereto; and the second reaction resulting in covalent bond formation between the
• the polymer substrate employed, and the cyclic, metal ion coordinating ligand group in the reactive species employed in the process may be chosen among those already discussed above in connection with functionalized polymer substrates according to the invention.
• the bifunctional reagent employed will typically be a bifunctional organic compound, e.g. an organic compound having at least two reactive functional groups chosen among groups such as carboxyl, thiol, aminopropyl, halogen and epoxy.
• Epichlorohydrin is particularly useful as a bifunctional reagent for a number of types of polymer substrate, including polysaccharides and derivatives thereof having surface hydroxyl groups.
• the reactive species containing the cyclic, metal ion coordinating ligand group will suitably be one which gives rise to a functionality (in the resulting functionalized polymer substrate product) of one of the types described above.
• appropriate reactive species for use in the process of the invention will then include species containing one or more cyclic, metal ion coordinating ligand groups having a reactive ring -NH- group and being derived from heterocycles chosen among: triazacycloalkanes and -cycloalkenes or among tetraazacycloalkanes and -cycloalkenes, e.g. species containing one or more cyclic, metal ion coordinating ligand groups having a reactive ring -NH- group and being derived from heterocycles chosen among: ,4,7-triazacyclononane;
• agaroses and agarose derivatives are well suited as polymer substrates in the context of the above-described process according to the invention.
• a well-suited bifunctional reagent will then be epichlorohydrin, and it may be advantageous in this connection to further incorporate a reducing agent, such as sodium borohydride, in the reaction mixture when reacting the polymer substrate with epichlorhydrin.
• the scope of the present invention further encompasses functionalized polymer substrates obtained or obtainable by a process as described above for preparing a functionalized polymer substrate.
• the scope of the present invention also encompasses a process for preparing a functionalized polymer substrate which is in accordance with the invention, and which further comprises a metal ion coordinated to at least one of the cyclic, metal ion coordinating groups therein, the process comprising contacting a functionalized polymer substrate according to the invention with an aqueous solution of an inorganic salt [e.g. a nitrate, halide (fluoride, chloride, bromide or iodide), sulfate, perchlo- rate, tetrafluoroborate, hexafluorophosphate or phosphate salt] or organic salt [e.g.
• an inorganic salt e.g. a nitrate, halide (fluoride, chloride, bromide or iodide), sulfate, perchlo- rate, tetrafluoroborate, hexafluorophosphate or phosphate salt
• organic salt e.
• a metal ion-containing functionalized polymer substrate obtained or obtainable by such a process is also within the scope of the present invention.
• metal ion coordinating (chelating) ligands having strong affinity for hard metal ions (and metal ions of borderline hardness) An important and valuable application of functionalized polymer substrates as defined in the context of the present invention is the use of a metal ion containing embodiment thereof in the purification of a protein, the protein in question being in the form of a fusion protein wherein the protein of interest is fused at its amino or carboxy terminus to an oligopeptide "tag", such as an Asp-containing oligopeptide according to the invention.
• oligopeptide 'tag such as an Asp-containing oligopeptide according to the invention
• the chelating ligands serve two aims: (a) they fix the metal ion to a solid support and (b) they modulate the metal affinity binding and thus the strength and affinity specificity of the adsorption centre.
• the chelating ligand should form stable complexes with the metal ions so that no metal ions are released into the solvent phase or transferred to the biomolecules during adsorption and desorption of these molecules. At the same time it should also leave at least one, and preferably two or more coordination sites of the metal ion available for protein binding.
• ligands based on the macrocycles cyclen and tacn, and containing carboxymethyl or phosphonomethyl pendant arms, have been synthesised on the basis of published methods 2"4 .
• These ligands are 1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane (D03A), 1 ,7, ⁇ bis(phosphonomethyl)- 1 ,4,7,10-tetraazacyclododecane (D02P), 1 ,4,7-tris(phosphonomethyl)-1 ,4,7,10-tetraazacyclodo- decane (D03P), 1-(carboxymethyl)-1,4,7-triazacyclononane (T1A), 1,4-bis(carboxymethyl)-1,4,7- triazacyclononane (T2A) and 1,4-bis(phosphonomethyl)-1,4,7-triazacyclononane (T2P).
• D03A 1,
• a secondary amine group enables attachment to an activated gel (polymer substrate), generating the IMAC support (functionalized polymer substrate).
• a maximum coordination number of 10 for calcium has been reported, coordination sites will be available for protein binding.
• the three acrocycles D02P, D03A and D03P were prepared by literature methods 2"4 and characterised by NMR and mass spectroscopy (MS). Preparation of D03A and D03P was achieved in high yields, and scaling-up of the reaction to obtain larger quantities (e.g. gram quantities or more) was successful.
• Scheme 3a ORTEP plot of D03P
• Scheme 3b Space-filling model of D03P
• Tacn 1 A Tacn2A Tacn2P
• the first precursor for the cyclam synthesis (N,N',N",N'"- tetratosyI-1, 5,8,12-tetraazadodecane) was synthesised using two different methods 7 ' 8 . Both methods led to products with identical NMR spectra and melting points. The structure was also confirmed by mass spectroscopy. Cyclam-derived macrocycles containing two or three carboxymethyl and two or three phosphonomethyl arms, respectively (Scheme 5), can be synthesized in a manner analogous to that described for the tacn and cyclen molecules, and function as 12-membered ring analogues of tacn and cyclen systems with carboxymethyl and phosphonomethyl pendant arms as detailed above.
• the derivatized cyclam ligands may also be used for the complexation of larger metal ions, such as calcium or magnesium.
• Immobilization (covalent attachment) of ligands to polvmer substrate (matrix) and of metal ions to immobilized ligand uses immobilized metal complexes, produced by binding (complexation) of metal ions to chelating ligands attached to a chromatographic matrix or support, to capture proteins containing specific metal-binding sites. The adsorption of these proteins is based on the coordination interaction between the immobilized metal ion and elec- tron- donor groups from the protein surface.
• the generation of an immobilized metal complex typically involves several steps.
• the chelating ligand is firstly attached to a chromatographic matrix (polymer substrate) which has previously been activated using a difunctional reagent, e.g. epichlorohydrin or 1,4- butanediol diglycidyl ether (bisoxirane).
• a difunctional reagent e.g. epichlorohydrin or 1,4- butanediol diglycidyl ether (bisoxirane).
• the second step involves the complexation of a metal ion to the immobilized chelating ligand, normally by treatment with an aqueous solution of a salt (e.g. chloride or nitrate) of the metal ion in question.
• a salt e.g. chloride or nitrate
• the resulting metal chelate complex is able to interact with a protein molecule, dissolved in a liquid mobile phase, such that electron-donor groups of amino acid residues situated at the surface of the protein (e.g. an imidazole group of a histidine residue) displace weakly coordinated solvent ligands (e.g.H 2 0) and form a coordinative bond with the immobilized metal ion.
• electron-donor groups of amino acid residues situated at the surface of the protein e.g. an imidazole group of a histidine residue
• weakly coordinated solvent ligands e.g.H 2 0
• the elution of the bound protein is achieved by reducing the pH of the mobile phase, or alternatively by using competitive ligands which displace the protein from the coordination site on the metal ion.
• the selectivity of IMAC separations can be influenced by the choice of metal ion, chelating ligand and/or solvent conditions, or by modification of the target protein.
• Each of the ligands in question is designed to contain a free amine group for attachment to a solid support matrix.
• the resulting immobilised (im) ligands (/m-D03P, /m-D03A) have been tested for their ability to bind to calcium ions.
• the macrocycles may be immobilized on SepharoseTM using the standard epoxy-activation method previously described.
• the first step involves the treatment of the SepharoseTM gel (polymer substrate) with epichlorohydrin under basic conditions to produce an epoxy-activated gel.
• the attachment of the ligands is then achieved through reaction of the nucleophilic secondary amine group in the ring of the ligand with the electrophilic epoxide surface group of the epoxy-activated gel.
• the reaction with the epoxy group introduces a spacer group that allows the immobilised ligand more conformational freedom to interact with the protein.
• the amount of immobilised ligand on the matrix can be calculated by nitrogen-analysis. As shown in Table 1 below, the highest surface coverage (ligand density) is obtained with immobilization of cyclen per se.
• the D03A ligand be- came immobilized to a reasonable level (optimum ca. 300 ⁇ mol/g dry gel), whereas the D03P ligand did not become immobilized as well. This may have been due to longer storage of the epoxy-activated gel prior to the ligand immobilization for the D03P.
• the low ligand coverage may be due to the greater steric bulk of the ligand preventing access to all epoxy groups available for interaction.
• the immobilisation of the metal ions was achieved by incubating the gels with solutions of the corresponding metal-ion chlorides for a period of one hour at room temperature. To ensure full deprotona- tion of the acid groups of the pendant arms of the /rrHigands, the pH of the metal-ion solutions was raised to pH 10 (except in the case of Fe 3+ , in order to avoid formation of hydroxo or oxo species). The amount of metal ion bound was determined by atomic adsorption spectroscopy (AAS) measurements, summarised in Table 2 below.
• AAS atomic adsorption spectroscopy
• the immobilisation of Ca 2+ ions was thus achieved by incubating the gels (/m-D03A, im-D03P) with CaCI 2 solution (pH 10) for a period of one hour at room temperature.
• hard metal ions are those which parallel the proton with respect to their attachment to ligands, are small, are often of high charge, and which lack valence shell electrons which are easily distorted or removed; hard metal ions include, e.g., Ca 2+ , Mg 2+ , Mn 2+ , Cr 3+ , La 3+ and Fe 3+ .
• soft metal ions are large, of low charge or have valence shell electrons which are easily distorted or removed; soft metal ions include, e.g., Cu + , Ag + and Cd 2+ .
• Metal ions whose properties place them on the borderline between hard and soft - i.e. are of "borderline hardness" - include, e.g., Zn 2+ , Fe 2+ , Co 2+ , Ni 2+ and Cu 2+ .
• Hard metal ions have the strongest affinity for hard Lewis bases.
• amino acid residues in proteins containing carboxylate groups can bind strongly to hard metal ions, such as Ca 2+ .
• a specific fusion tag 20 is required.
• These hard metal ion binding tags are ideally short peptide tags that can be introduced into the protein using recombinant genetic techniques.
• specific Ca 2+ -binding tags naturally occurring Ca 2+ -binding proteins were analysed for their amino acid sequence involved in Ca 2+ binding. These sequences bind Ca 2+ with high affinity and as such have the potential to provide a basis for tag design. Examples of such proteins are the calcium-binding proteins calmodulin (CaM), troponin C (TnC), and parvalbumin (Parv). All these proteins bind Ca 2+ with high affinities 13 :
• Scheme 7 shows the structure of calmodulin with the four calcium-binding sites. It is generally accepted that two of the four binding sites have lower affinity for Ca 2+ , and that the two other sites have slightly higher affinity for Ca 2+ .
• the structural studies show that the molecule has a "dumbbell" shape, with two globular ends connected by a long, exposed ⁇ -helix. Each end has two Ca 2+ -binding sites, each with a loop of 12 amino acid residues in which residues such as aspartic acid and glutamic acid residues (shown in bold print in Table 3, below) form electrostatic/coordinate bonds with calcium.
• the two Ca 2+ -binding sites in the carboxyl-terminal part of the molecule Domains III, IV
• the invention also encompasses variants of such sequences wherein one or more amino acid residues (e.g. a single amino acid residue) are replaced with (i.e. substituted by) a different amino acid residue, particularly with an amino acid residue or amino acid residues which is of similar chemical functionality to the the replaced amino acid residue.
• amino acid residues e.g. a single amino acid residue
• similar chemical functionality is intended to indicate that the replaced amino acid residue and the amino acid residue replacing it are closely related with respect to polarity, with respect to polarizability, with respect to number of acidic and basic substituents and/or with respect to structural analogy or homology.
• Oligopeptides comprising one or more of the amino acid sequences shown in Scheme 8, and/or one or more substitution variants of such sequences, also constitute an aspect of the present invention.
• the 'tag" sequences in question (or substitution variants thereof) may be identical or different.
• a peptide (oligopeptide) 'tag may comprise, in addition to one of the amino acid sequences shown above, or a substitution variant thereof as described above, one or more additional amino acid residues, such as from 2 to 6 or more additional amino acid residues, attached at the C- or N-terminal end of the oligopeptide "tag” (e.g. at the C- or N-terminal end of one of the eight se- quences (Tag 1 - Tag 8) shown in Scheme 8.
• oligopeptide 'tag e.g., a C-terminally located oligopeptide 'tag
• the oligopeptide "tag” e.g.
• one of those listed above in Scheme 8) can be simultaneously fused to two molecules of the protein or polypeptide of interest, or alternatively to two different proteins or polypeptides of interest, at their amino- or carboxy- terminus, respectively, thereby forming a new fusion protein structure whereby the oligopeptide 'tag" is located at an e ⁇ cto-position (i.e. at an internal position) linking the two molecules of the protein(s) or polypeptide(s) of interest.
• coupling reagents HABt, HBTU, DIPEA
• the deprotection and coupling step is re- peated until the final crude peptide is obtained.
• Standard TFA cleavage methods may be employed to cleave the product from the resin and remove side-chain protecting groups from the crude peptide.
• the crude peptides were purified by preparative Reverse-Phase High-Pressure Liquid Chromatogra- phy (RP-HPLC), e.g. using gradient elution with increasing concentrations of acetonitrile.
• Still further aspects of the invention include the following:
• fusion proteins of the type in question include fusion proteins wherein two molecules of a protein or polypeptide of interest, or alternatively two different proteins or polypeptides of interest, are attached simultaneously at their amino- or carboxy- terminus, respectively, to one and the same oligopeptide (i.e.
• oligopeptide 'tag an oligopeptide accord- ing to the invention, so as to form a fusion protein structure in which the oligopeptide (i.e. the 'tag") is located at an encfo-position (i.e. an internal position) linking the two molecules of the protein(s) or polypeptide(s) of interest; in fusion protein structures of this type, the amino acid sequence of the oligopeptide 'tag" may suitably be flanked by one or more enzymatic or chemical cleavage sites;
• polynucleotide construct such as a vector, encoding such a polypeptide
• a polypeptide obtainable by cultivating a host cell (e.g. a prokaryote such as Escherichia coli) comprising such a polynucleotide construct in an appropriate growth medium under conditions allowing expression of the polypeptide, and recovering the polypeptide from the culture medium;
• a host cell e.g. a prokaryote such as Escherichia coli
• a host cell e.g. a prokaryote cell (e.g. a strain of Escherichia coli), comprising such a polynucleotide construct; and
• a method for producing a polypeptide of the type in question comprising cultivating a host cell of the type in question in an appropriate growth medium under conditions allowing expression of the polypeptide, and recovering the polypeptide from the culture medium;
• Yet another aspect of the invention relates to a method for purifying a protein of interest, the method comprising the steps of:
• a protein sample which contains: a polypeptide which is a fusion protein comprising the protein of interest fused at its amino terminus or carboxy terminus to at least one oligopeptide (i.e. oligopeptide 'tag") according to the invention (i.e. a fusion protein of one of the types already mentioned above, including a fusion protein in which the oligopeptide (i.e. the 'tag") is situated in an endo position as already discussed); and other (extraneous) proteins; with a metal ion-containing functionalized polymer substrate according to the invention under conditions whereby the polypeptide (fusion protein) binds to the metal ion-containing functionalized polymer substrate so as to form a complex therewith;
• oligopeptide i.e. oligopeptide 'tag
• the latter method may further comprise a step wherein the oligopeptide (the 'tag") is cleaved from the polypeptide or protein of interest, e.g. by chemical means or by means of an enzyme, e.g. an endo- or exo-peptidase.
• the invention also encompasses a purified protein obtained or obtainable by the latter method.
• Affinity tags are short fragments of DNA that code for an amino acid sequence which has a strong binding affinity for a metal ion. These tags (c-DNA) are ligated (joined) to the c-DNA of the target recombinant protein at the N- or C- terminus.
• the resulting tagged fusion protein can be expressed (produced) and subsequently purified from crude cell extract using IMAC systems since the attached tag binds selectively or specifically to the immobilized metal ions. Elution of the fusion protein can be achieved by applying a continuously decreased pH gradient. Alternatively, a chelating agent such as EDTA can be added to the mobile phase to elute the protein. If required, the affinity tag can be removed from the target protein (post purification) using chemical or enzymatic methods.
• Polypeptides or proteins which are of relevance in relation to the purification methodology taught in the context of the present invention include the following: mammalian proteins, such as, e.g., growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipopro- teins; ⁇ -1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calci- tonin; luteinizing hormone; glucagon; clotting factors, such as Factor VII (including Factor Vila), Factor VIII, Factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or tissue-type plasmino- gen activator (t-PA); bombazine; thrombin; tumor necrosis factor
• mammalian proteins such as,
• polynucleotide denotes a single- or double-stranded polymer of deoxy- ribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
• Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules.
• the length of a polynucleotide molecule is given in terms of nu- cleotides (abbreviated “nt”) or base pairs (abbreviated "bp").
• nt nu- cleotides
• bp base pairs
• double-stranded molecules When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double- stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20.pt in length.
• host cell denotes any cell, including a hybrid cell, in which heterolo- gous DNA can be expressed.
• Typical host cells include, but are not limited to, bacterial cells, insect cells, yeast cells and mammalian cells, including human cells, such as BHK, CHO, HEK, and COS cells.
• the term 'Vector denotes any nucleic acid entity capable of amplification in a host cell.
• the vector may be an autonomously replicating vector, i.e. a vector that exists as an ex- trachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
• the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. The choice of vector will often depend on the host cell into which it is to be introduced.
• Vectors include, but are not limited to plasmid vectors, phage vectors, viruses or cosmid vectors.
• Vectors usually contain a replication origin and at least one selectable gene, i.e. a gene that encodes a product which is readily detectable, or the presence of which is essential for cell growth.
• selectable gene i.e. a gene that encodes a product which is readily detectable, or the presence of which is essential for cell growth.
• amino acid residues are represented using abbreviations approved by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN). With respect to amino acids, those represented by the following abbreviations are in the naturally occurring L-form. Further, the left and right ends of an amino acid sequence of a peptide are, respectively, the N- and C-termini unless otherwise specified.
• TacnlA or T1 A 1 -(carboxymethyl)-l ,4,7-triazacyclononane
• 1,4,7,10-Tetraazatricyclotridecane (1 ; 4g, 22.03mmol) was cooled down to 4°C, and an EtOH/H 2 0 solution (50ml, chilled to -20°C) was added. The resulting reaction mixture was allowed to slowly warm to room temperature and then stirred under nitrogen for 12 hours. The reaction mixture was concentrated in vacuo, dissolved in acetonitrile (50ml), and concentrated again. This process was repeated three times to remove traces of H 2 0. The pale yellow oil was dried under vacuum at room temperature overnight to yield a white hydroscopic solid.
• the toluene layer was extracted three times with 1M Na 2 C0 3 (50ml), followed by 0.8M HCI (1x25ml) and finally with H 2 0 (25ml).
• the aqueous layers were combined and the pH adjusted to 9.4 using Na 2 C0 3 .
• These combined layers were then extracted twice with dichloromethane (DCM; 50ml), and the DCM layers were combined and dried over Na 2 C0 3 .
• the organic layer was concentrated in vacuo to yield 3 as a viscous pale yellow oil.
• 1-formyl-1 ,4,7,10-tetraazacyclododecane (2g, 10mmol) and triethylphosphite (6g, 36.1 mmol, 20%excess) were placed in a round-bottom flask, and the flask was immersed in an ice bath.
• Paraformaldehyde (1g, 33mmol, 10% excess) was added in small portions over a period of 30 minutes. The mixture was then allowed to warm to room temperature, and stirred for 2 days at room temperature followed by one day at 40-50°C. The clear mixture was kept under high vacuum at 50°C for several hours to remove volatile impurities.
• the resulting crude phosphonate ester (5) was hydrolysed without further purification.
• SepharoseTM 6FF (500g) was washed extensively with water, suction dried and placed in a round- bottom flask. 2M NaOH (500ml) containing NaBH 4 (1.88mg/ml) was added, and the suspension was stirred for 2 hours at room temperature. Epichlorohydrin (300ml) was then added and the suspension stirred for 5 days. The resulting epoxy-activated gel was collected by vacuum filtration, washed copiously with water (5x500ml) and stored in 20% ethanol at 4°C until used for ligand immobilisation.
• a 0.2M solution of D03A (20 ml) was prepared and adjusted to pH 12 with 2M NaOH. This D03A solution was added to suction-dried epoxy-activated SepharoseTM 6FF (20g), and the reaction mixture was shaken for 4 days at room temperature. The resulting immobilised D03A gel (/m-D03A) was filtered off, washed extensively with water (5x50 ml) and stored in 20% ethanol at 4°C.
• D03P Due to the insolubility of D03P in water, a suspension of D03P in water (10 ml) was prepared and the pH adjusted to 12 with 2M NaOH. By thus increasing the pH, the D03P was found to dissolve. The resulting solution was diluted to a final volume of 20ml, and suction-dried epoxy-activated Sepha- roseTM (20g) was added. The suspension was incubated for 4 days at room temperature. After this time the immobilised D03P gel (/m-D03P) was collected by vacuum filtration and washed with 5x50ml of water. The gel was stored at 4°C in 20% ethanol.
• the solution was diluted to exactly 50 ml with H>0, and the Ca 2+ (or other metal ion) content determined by atomic absorption spectroscopy (AAS) using a Varian AA-1475 atomic absorption spectrometer at a metal-specific wavelength and working range (e.g. for Ca 2+ ions at a wavelength of 422.7nm and with a working range from 1 to 4 ppm).
• AAS atomic absorption spectroscopy
• the resin/peptide product was transferred to a plastic sinter with DMF, and washed with methanol (2x10 ml) and diethyl ether (10 ml). The crude resin/peptide product was dried in a desiccator overnight.
• RP-HPLC Reverse-Phase HPLC
• Detection was carried out using a Model 486 variable wavelength UV-detector connected to the Waters Millennium software computer. ⁇
• the peptides were purified by preparative RP-HPLC with different elution gradients (Table III, below) of Buffer A (0.1% TFA) to Buffer B [90% (v/v) acetonitrile/water, 0.1% TFA] over 1 hour with a flow-rate of 6ml/min, and with detection at a wavelength at 230 nm using a TSK- ODS-120T C-18 (300 x 21.55 mm) column from TOSOH (Tokyo, Japan).
• the fractions from the prepa- rative runs of RP-HPLC were collected with a Pharmacia (Frac-100) fraction collector from Pharmacia Biotech AB (Uppsala, Sweden), and freeze-dried overnight.
• the purity of the collected fractions was determined by analytical RP-HPLC using a TSK-ODS-120T C-18 (150 x 4.6 mm) column from TOSOH (Tokyo, Japan) with an elution gradient of Buffer B (0 - 85%) over 25 minutes with a flow-rate of 1 ml/min and with detection at 214 nm.
• the purified peptides obtained were further analysed by analytical RP-HPLC using a longer acetoni- trile gradient and the molecular weights confirmed by mass spectroscopy.
• peptides 1, 2, 3G, 4, 5 and 6G all have spectra corresponding to the calculated mass.
• the peptides 3R and 6R were found to contain a deletion product, peptide 6R with the methionine missing and peptide 3R with an arginine deletion.
• optimisation of the elution gradient is required whereby the gradient length is increased considerably.
• the molecular weights (MW) of the peptides were determined by Electrospray lonisation Mass Spec- troscopy (ESI-MS) using a Micromass platform (II) quadrupole MS with Electrospray source and Mass- lynx NT version 3.2 software (Micromass, Cheshire, UK).
• the peptides were dissolved in a 1 :1 mixture of 50% (v/v) acetonitrile/water and 3% (v/v) formic acid.
• the scan range was 0-2000 m/z, and samples were injected via a manual injector at a rate of 10 ⁇ l/min.
• target protein glutthione S-transferase, GST
• GST-tag fusion proteins was carried out at different scales, whereby single colonies of E. coli BL 21 host cells containing the recombinant GST-tag plasmids (Tag 1 to 6, untagged GST and vector only) were inoculated directly into 10 ml of 2xYT medium (16g/l Tryptone, 10 g/l yeast extract, 5 g/l NaCI, containing 100 ⁇ g/ml of Ampicillin). The cultures were incubated overnight at 37°C with vigorous shaking.
• the solutions were adjusted to a total volume of 30 ml, and a hen-egg-white lysozyme solution added (50 mg/ml, 1/100 of total volume). Following incubation of the solutions on ice for 10 min, solutions of MgCI 2 (2M, 1/1000 of total volume) and DNAse-l (10mg/ml, 1/1000 of volume) were then added, and the solutions were incubated again for 20 min on ice. Finally, the suspended cells were disrupted by sonication on ice with three short 30 sec burst with a 30 sec pause between each sonication. The lysate was separated from cell debris by centrifugation in a SS34 rotor at 13000xg for 20 min at 4°C. The supernatants were retained for purification.
• the 5x Sample Buffer consists of: 1.5M Tris-CI pH 8.8 1.5 ml Glycerol 2.5 ml SDS 0.5 g Bromophenol blue 2 mg Betamercaptoethanol 1.0 ml Water up to a final volurr le of 5.0 r
• the SDS-polyacrylamide gels were run in Tank Running Buffer (0.025M Tris, 0.192M glycine, 0.1% SDS, pH 8.3) using the Hoeffer Mini VE Vertical Electrophoresis System at a constant current of 20 mA per gel, until the dye-front reached the bottom of the resolving gel.
• the gels were stained using the silver staining method of Swain and Ross , as indicated below Silver stain protocol (per ae ⁇ )
• the gels were stained overnight using Coomassie stain, which stains the entire gel blue. On the following day the gel is de-stained, whereby only the protein bands retain the blue colour.
• the tags were introduced to the protein GST by recombinant DNA technology.
• GST was used as model protein because it is commercially available, easy to express, well characterised and purified easily from cell lysates using Glutathione SepharoseTM 4B.
• Expression of the tagged-GST protein molecules is achieved using host bacterial cells (Escherichia coli BL21). These cells were induced with IPTG to express the tagged-GST proteins.
• the successfully purified tagged GST proteins from the contaminating host cell proteins were analysed by SDS-Page. As can be seen in Scheme 9, below, the starting material (SM) and flow through (FT) contain a lot of contaminating host proteins of various sizes.
• the elution fraction from the purification (E) shows that for each tagged-GST, there is a band corresponding to the theoretical sizes ( ⁇ 26kDa), and that the eluted protein is relatively pure.
• Tag1- GST, Tag2-GST, Tag4-GST and Tag6-GST have a smaller band below the main band that may correspond to truncated tagged-GST formed via the action of host cell proteases.

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